Fused DNA sequence, fused protein expressed from said fused DNA sequence and method for expressing said fused protein

ABSTRACT

Disclosed are a fused DNA sequence which comprises a DNA sequence of a heat-resistant protein, fused directly or indirectly to a DNA sequence coding a selected protein or peptide, a fused protein expressed from the fused DNA sequence, and a method for expressing the fused protein.

BACKGROUND OF THE INVENTION

This invention relates to expression of a fused protein, morespecifically to a fused DNA sequence including a DNA sequence coding aheat-resistant protein, a fused protein expressed by said fused DNAsequence, and a method for expressing said fused protein.

Progress in genetic engineering has enabled analysis of a protein whichhas been purified from a natural substance, at a genetic level andartificial amplification of a desired protein (Itakura et al., Science,vol. 198, p. 1056 (1977)). By application of a DNA sequence to whichthio-redoxin (hereinafter referred to as “TRX” in the specification)(International Provisional Patent Publication No. 507209/1993) orglutathione-S-transferase (hereinafter referred to as “GST” in thespecification) (International Provisional Patent Publication No.503441/1989) which has been invented thereafter is fused, even a proteinwhich is inherently expressed with difficulty can be expressed, and atechnique of expressing a fused protein has been used widely.

TRX and GST can be applied to fusion and expression of various proteinswhich are expressed with difficulty, but even in GST which has beenessentially used for the purpose of expressing a soluble fused protein,a fused protein becomes insoluble depending on a protein to be fused sothat productivity is lowered, or a fused protein to which TRX is fusedmay have a problem that a nonspecific reaction is liable to occur.Therefore, it has been desired to provide a fused protein having furtherexcellent operatability and productivity.

SUMMARY OF THE INVENTION

Thus, an object of the present invention is to provide a novel fused DNAsequence having excellent operatability and productivity for expressinga desired protein or peptide, a fused protein expressed from said fusedDNA sequence, and a method for expressing the fused protein using saidfused DNA sequence.

The present inventors have studied intensively in order to solve theproblems in the art and consequently found that when a DNA sequencecoding a selected protein or peptide and a DNA sequence coding aheat-resistant protein are fused directly or indirectly and a fusedprotein is expressed from the resulting fused DNA sequence, theproductivity of the desired protein or peptide is raised, and said fusedprotein has heat resistance to make a purification step simple and easy,to accomplish the present invention.

That is, the present invention relates to a fused DNA sequencecomprising a DNA sequence coding a heat-resistant protein or peptide,fused directly or indirectly to a DNA sequence coding a selected proteinor peptide, a fused protein expressed by said fused DNA sequence, and amethod for expressing the fused protein using said fuse-DNA sequence.

The fused protein of the present invention has high solubility and canmaintain even heat resistance derived from heat-resistant protein genes.Because of such a characteristic of the fused protein, when the fusedprotein is purified, unnecessary substances can be removed simply andeasily by heat treatment so that the fused protein can be obtained withgood yield.

In the case of TRX derived from Escherichia coli and GST derived fromSchistosoma japonicum, which have been widely used as a fused protein,Escherichia coli and Schistosoma japonicum can live in bodies of mammalsand other creatures so that when a fused protein using TRX or GST isused as an antigen of an immunoreaction, a nonspecific reaction due toEscherichia coli or Schistosoma japonicum might be caused. To thecontrary, the great characteristic of the fused protein of the presentinvention resides in that a heat-resistant protein derived from athermophilic bacterium which cannot live in living bodies of mammals andother creatures is used so that even when the fused protein of thepresent invention is used as an antigen of an immunoreaction, anonspecific reaction derived from the fused protein is caused withdifficulty.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a detailed view of an expression vector pW6A.

FIG. 2 is a detailed view of an expression vector pWF6A.

FIG. 3 is a graph showing the reactivity of a fused protein and anegative specimen.

FIG. 4 is a graph showing the reactivity of a HTLV-I-fused protein and apositive specimen.

FIG. 5 is a graph showing the reactivity of a HTLV-II-fused protein anda positive specimen.

FIG. 6 is a graph showing the reactivity depending on concentration of aHTLV-I-fused protein.

FIG. 7 is a graph showing the reactivity depending on concentration of aHTLV-II-fused protein.

FIG. 8 is a graph showing the activity of a fused protein in asupernatant subjected to heat treatment.

FIG. 9 is a graph showing the activity of a fused protein ofprecipitates subjected to heat treatment.

FIG. 10 is a view showing the activity of a fused protein after heattreatment and purification.

FIG. 11 is a detailed view of an expression vector pW6AK.

FIG. 12 is a view showing the activity of a fused protein after heattreatment and purification.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following, the present invention is explained in detail.

The DNA sequence coding a heat-resistant protein of the presentinvention means a DNA sequence coding a protein which is not thermallydenatured even at 55° C. or higher, preferably 75° C. or higher. As aspecific phenomenon of thermal denaturation, there may be mentionedinactivation or insolubilization of a protein. As the DNA sequencecoding a protein which is not thermally denatured at 55° C. or higher,there may be mentioned, for example, a DNA sequence possessed by athermophilic bacterium which can live at 55° C. or higher. From theproperties of an expressed protein and easiness of post-treatment, it ispreferred to use a DNA sequence possessed by the so-called highlythermophilic bacterium which can live at 75° C. or higher. As the highlythermophilic bacterium, there may be mentioned, for example,Thermophilus, Sulfolobus, Pyrococcus, Thermotoga, Pyrobaculum,Pyrodictium, Thermococcus, Thermodiscus, Metanothermus and Metanococcus(FEMS. MICRO. BIOL. REV., Vol.75, pp.117-124 (1990), ANU. REV.MICROBIOL., Vol.47, pp.627-653 (1993)). As the heat-resistant protein,there may be mentioned, for example, adenylate kinase derived from aSulfolobus bacterium (Sulfolobus acidocaldalius Adenylate kinase: Arch.Biochem. Biophys., Vol.207, pp.405-410 (1993)) (hereinafter referred toas “AK” in the specification), DNA polymerase derived from aThermophilus bacterium, ferredoxin derived from a Pyrococcus bacterium(Pyrococcus furiosus Ferredoxin: Biochemistry, Vol.31, pp.1192-1196(1992)) (hereinafter referred to as “FDX” in the specification),glucosidase derived from Pyrococcus furiosus bacterium (Pyrococcusfuriosus Glucosidase), rubredoxin derived from Pyrococcus furiosusbacterium (Pyrococcus furiosus Rubredoxin: Biochemistry, Vol.30,pp.10885-10895 (1991)) glutamate dehydrogenase derived from Pyrococcusfuriosus bacterium (Pyrococcus furiosus Glutamate dehydrogenase: Gene,Vol.132, pp.189-197 (1988)), glyceraldehyde phosphate dehydrogenasederived from Metanothermus fervids bacterium (Metanothermus fervidsGlyceraldehyde 3-phosphate dehydrogenase: Gene, Vol.64, p.189-197(1988)), glutamate synthetase derived from Metanococcus volate bacterium(Metanococcus volate Glutamate synthetase: Res. Microbiol., Vol.140,pp.355-371 (1989)), L-lactate dehydrogenase derived from Thermotogamaritina bacterium (Thermotoga maritina L-lactate dehydrogenase: Eur. J.Biochem., Vol.216,; pp.709-715 (1993)) and elongation factor derivedfrom Thermococcus celer bacterium (Thermococcus celer Elongation FactorI-alpha: Nucleic acid res. Vol.18, p.3989 (1990)), but theheat-resistant protein coded by the DNA sequence of the presentinvention is not limited thereby. DNA coding the heat-resistant proteinof the present invention can be purified from these highly thermophilicbacteria, but it can be also synthesized based on a known DNA sequence.For synthesis of DNA of the heat-resistant protein, a known techniquesuch as a β-cyano-ethylphosphoamidite method (Sinha et al., NucleicAcids Bos., Vol.12, p.4539 (1984)) and a method described in Letsinger,R. L. et al., J. Am. Chem. Soc., vol. 88, p. 5319 (1966) may be suitablyused. In Examples each of which is an embodiment of the presentinvention, DNA's of FDX derived from Pyrocuccus bacterium and AK derivedfrom Sulfolobus bacterium having amino acid sequences shown in SEQ IDNO: 2 and 4, respectively, are synthesized by theβ-cyanoethylphosphoamidite method. DNA sequences synthesized are shownin SEQ ID NO: 1 and 3, respectively.

The DNA sequence coding a selected desired protein or peptide of thepresent invention is not limited to a particular DNA sequence. Any DNAsequence can be used so long as it is a DNA sequence coding a protein orpeptide which is desired to be expressed as a fused protein. The presentinvention is particularly useful when a necessary expression amount of aselected desired protein or peptide can be obtained with difficulty byDNA itself coding said protein or peptide.

The fused DNA sequence of the present invention can be fused by using aknown method such as a ligation method and a linker ligation method.When fusion is carried out, the DNA sequence of a selected desiredprotein or peptide and the DNA sequence of the heat-resistant proteinmay be fused directly or may be fused indirectly, if necessary. In thecase of indirect fusion, a linker,sequence is inserted between the DNAsequence coding a desired protein or peptide and the DNA sequence codingthe heat-resistant protein. As said linker sequence, there can be used asequence coding a polypeptide for bonding a desired protein or peptideand the heat-resistant protein to each other and a sequence coding apolypeptide which can be cleaved or digested selectively by a knownchemical method or enzymatic method. When the linker sequence isinserted between the DNA sequence coding a desired protein or peptideand the DNA sequence coding the heat-resistant protein, only a selecteddesired protein or peptide portion can be also purified by, after thefused protein is expressed, cleaving or digesting the linker sequence byusing a chemical means such as bromocyan or an enzymatic means such asthrombin or a factor Xa.

In order to express the fused protein of the present invention, a commontechnique of genetic engineering can be used. For example, the fused DNAsequence of the present invention is inserted into a vector which issuitable for expression, said vector is introduced into a culture host,and expression of the fused protein is induced. After the host is grownby culture or the like, sonication of the host and purification such asa column operation are carried out to obtain a desired fused protein orpeptide. Host cells to be used may be any cells such as bacterial cells,eucaryotic cells and mammal cells so long as they are cells which canexpress a foreign protein or peptide, and there may be mentioned, forexample, Escherichia coli, yeast, Bacillus subtilis, Baculo virus andCOS cells.

The fused protein of the present invention may be used as such as afused protein, or a desired protein or peptide portion thereof obtainedby separation and purification may be used.

EXAMPLES

The present invention is described in detail by referring to Referenceexamples and Examples.

Example 1 Preparation of FDX-expressing Vector pWF6A

By using 8 primers of 53 mer prepared based on a known DNA sequence ofPyrococcus furiosus FDX by using a DNA synthesizer (Model 392, tradename, manufactured by PERKIN ELMER Co.), genes of Pyrococcus furiosusFDX were synthesized by the assemble PCR (polymerase chain reaction)method. In the assemble PCR method, a Taq polymerase (produced by ToyoboCo.) was used, and the total base number of 248 bp was amplified underconditions of 30 cycles of 94° C.−1 minute, 55° C.−1 minute and 72° C.−1minute. A restriction enzyme NdeI site was added to 5′-end, arestriction enzyme EcoRI site was added to 3′-end, and a thrombin-cutsite was added to C terminal. This fragment was integrated into the NdeIand EcoRI sites of 4.6 Kb of a pW6A vector prepared from pGEMEX-1 (tradename, produced by Promega Co.) and pGEX-2T (trade name, produced byPharmacia Biotec Co.) to prepare pWF6A as a vector expressing FDX. Adetailed view of pW6A is shown in FIG. 1, and a detailed view of pWF6Ais shown in FIG. 2. pWF6A contains, at the NdeI and EcoRI sites, genesof a fused protein comprising 96 amino acids including 67 amino acidsderived from FDX, 10 amino acids derived from a thrombin-cleaved siteand 19 amino acids derived from multi cloning site of pW6A. The basesequence of the inserted fragment was confirmed by a DNA sequence kit(trade name: Sequenase kit Ver. 2.0, produced by Amersham United StatesBiochemical Co.). DNA sequence of the FDX inserted into pW6A and aminoacids sequence coded by said sequence are shown in SEQ ID NO: 1 and SEQID NO: 2, respectively, and DNA sequence of the pW6A is shown in SEQ IDNO: 5. In the sequence table, ATG of the restriction enzyme site NdeI isshown as 1 and sequences before the stop codon of a multi-cloning siteare shown. pWF6A was introduced into host Escherichia coli and thencultured for 2 hours in a medium (hereinafter referred to as “the LBmedium” in the specification) containing 1% of bactotryptone, 0.5% ofyeast extract, 1% of sodium chloride and 50 μg/ml of ampicillin andhaving pH 7.5. Thereafter, 1 mM isopropyl thio-galactopyranoside(hereinafter referred to as “IPTG” in the specification) was addedthereto, and the mixture was cultured for 2 hours to induce expression.10 mM Tris-hydro-chloride having pH 7.5 and 1 mMethylenediaminetetraacetic acid (hereinafter abbreviated to as “EDTA” inthe specification) (in the following, this buffer is referred to as “aTE buffer” in the specification) were added to the precipitates ofEscherichia coli, the precipitates were sonicated, and 15% sodiumdodecylsulfate-polyacrylamide gel electrophoresis (hereinafter referredto as “SDS-PAGE”) according to the Laemmli method was carried out. ByCoomassie brilliant blue staining (hereinafter referred to as “CBBstaining” in the specification), a band was confirmed at about 22 Kda,and FDX of Pyrococcus furiosus forming a dimer was recognized.

Example 2 Purification of FDX

pWF6A prepared in Example 1 was introduced into host Escherichia coliand then cultured under conditions of using the LB medium at 37° C. Bypreculture, a concentration of Escherichia coli in a culture broth wasmade to have such turbidity that absorbance at a wavelength of 600 nmwas about 1.0, 1 mM IPTG was added thereto to induce expression. Afterthe mixture was cultured for 3 hours, centrifugation was carried out torecover Escherichia coli. 200 ml of a 50 mM Tris-hydrochloride buffer(hereinafter referred to as “the Tris buffer” in the specification)having pH 8.0 was added to recovered Escherichia coli, followed bysonication treatment under ice cooling. After centrifugation, theexpressed fused protein was recovered in the supernatant as a solublecomponent. When this supernatant was subjected heat treatment at 85° C.for 15 minutes, about 80% of the Escherichia coli protein was thermallydenatured and precipitated, and 90% or more of FDX was recovered in thecentrifugation supernatant after the heat treatment.

This supernatant was purified by ion exchange using a QFF anion exchangecolumn (trade name, manufactured by Pharmacia Biotec Co.) equilibratedwith the Tris buffer. When the supernatant was eluted by a columnequilibrated buffer containing sodium chloride, FDX was recovered at aconcentration of about 0.3 M sodium chloride-eluted fraction. Then, thisFDX fraction was purified by using a RESOURCE RPC column (trade name,manufactured by Pharmacia Biotec Co.) equilibrated with 20 mM sodiumhydroxide. When the fraction was eluted by acetonitrile, purified FDXwas recovered at a concentration of about 10% acetonitrile-elutedfraction.

Reference Example 1 Purification of TRX

pWT8A prepared as a vector expressing TRX in the same manner as in pWF6Aprepared in Example 1 was introduced into host Escherichia coli and thencultured under conditions of using the LB medium at 37° C. After thesame induction of expression as in Example 1 was carried out,Escherichia coli was recovered by centrifugation. An osmotic shock wasgiven to recovered Escherichia coli, and TRX existing at a periplasmicfraction was extracted. Extracted TRX was subjected to firstpurification by using a RESOURCE RPC column (trade name, manufactured byPharmacia Biotec Co.) equilibrated with 20 mM sodium hydroxide. When TRXwas eluted by acetonitrile, TRX was recovered at a concentration ofabout 10% to 20% acetonitrile-eluted fraction. Recovered TRX wasdialyzed to 4 M guanidine hydrochloride and then subjected to secondpurification by using the reverse phase column under the sameconditions. Similarly as in the first purification, purified TRX wasrecovered at a concentration of about 10% to 20% acetonitrile-elutedfraction.

Example 3 Specificity Test of FDX and TRX by the Western Botting Method

An anti-Escherichia coli antibody was supposed as a non-specificreaction substance, and the reactivities of FDX purified in Example 2and TRX purified in Reference example 1 were examined.

A SDS-solubilized material of Escherichia coli DH5α, a supernatant ofEscherichia coli DH5α sonicated and a SDS-solubilized material ofEscherichia coli to which a pW50 vector (made by Fuji Rebio) wasintroduced were used as immunogen and immunized to 3 rabbits to preparethe total 9 kinds of the respective anti-Escherichia coli rabbit serums.FDX purified in Example 2 and TRX purified in Reference example 1 weresubjected to SDS-PAGE according to the Laemmli method and thentransferred to nitrocellulose membranes. After blocking the proteinportion adsorbed to the nitrocellulose membranes with 1% skim milkdissolved in PBS, the western blotting method was carried out by usingthe above 9 kinds of the anti-Escherichia coli rabbit serums diluted 500times, respectively, as primary anti-bodies, and using a peroxidase(hereinafter referred to as “POD” in the specification)-labeledanti-rabbit antibody as a secondary antibody. For coloring,4-chloro-1-naphthol and hydrogen peroxide were used. At the portioncorresponding to the molecular weight of FDX, no substance reacting withthe anti-Escherichia coli rabbit antibody was confirmed, but at theportion corresponding to the molecular weight of TRX, among 9 kinds ofthe anti-Escherichia coli rabbit serums, 6 kinds of the serums in whichthe supernatant of Escherichia coli DH5α sonicated and theSDS-solubilized material of Escherichia coli into which the pW50 vectorwas introduced were used as immunogen were reacted, respectively.

In the same manner as described above, the western blotting method wascarried out by 25 samples of human specimen HTLV-I/II mix panel 204serums (trade name, produced by Boston Biomedica Co.) diluted 50 times,respectively, as primary antibodies, and using POD-labelled anti-humanIgG as a secondary antibody. Reactivities at sites where FDX wastransferred was not confirmed; but the reactions of 2 samples among 25samples at sites where TRX was transferred were confirmed. The resultsare shown in Table 1.

TABLE 1 Intensity of reaction (+, −) by western blotting Specimen No.FDX TRX PRP-204-01 − − PRP-204-02 − − PRP-204-03 − − PRP-204-04PRP-204-05 − − PRP-204-06 PRP-204-07 − − PRP-204-08 PRP-204-09 − −PRP-204-10 PRP-204-11 − − PRP-204-12 − + PRP-204-13 − − PRP-204-14PRP-204-15 − − PRP-204-16 PRP-204-17 − − PRP-204-18 PRP-204-19 − −PRP-204-20 − − PRP-204-21 PRP-204-22 − − PRP-204-23 − + PRP-204-24 − −PRP-204-25 − − +: positive, −: negative

Example 4 Specificity Test of FDX and TRX by the ELISA Method UsingHuman Specimens

On ELISA plates (produced by Becton Deckinson Co.) were sensitized each50 μl of 25 μg/ml of FDX purified in Example 2 and TRX purified inReference example 1, respectively.

After blocking the protein portion adsorbed onto wells of the ELISAplate with 1% skim milk, a specificity test according to the ELISAmethod was carried out by using the human specimens produced by BostonBiomedica Co. diluted 500 times used in Example 3 as primary antibodiesand POD-labelled anti-human IgG as a secondary antibody. For coloring,ABTS and hydrogen peroxide were used. The measurement results were shownby difference between absorbances at a wavelength of 405 nm and awavelength of 492 nm (difference between absorbances was described asA405/492 nm). In the reactions with the specimens, whereas there was nospecimen exceeding twice of a blank in the case of FDX, the specimensexceeding twice of a blank were confirmed in 6 samples among 25 samplesin the case of TRX. FDX derived from Pyrococcus furiosus was differentfrom TRX derived from Escherichia coli in that neither nonspecificreaction nor cross reaction derived from Escherichia coli wasrecognized. The results are shown in FIG. 3.

Example 5 Expression of FDX-Fused HTLV-I p19-Fused Protein and FDX-fusedHTLV-II p19-fused Protein

From infected cell lines expressing HTLV-I and HTLV-II, genomic DNA wasextracted by the method of Molecular Cloning by J. Sambrook et al. Next,by using a primer to which EcoRI and BamHI sites were added, the PCRmethod was carried out in the same manner as in Example 1 to obtainabout 400 bp of p19DNA fragments in the respective gag regions. Thesefragments were integrated into pWF6A to prepare pWFIP19 as a vectorexpressing p19 of HTLV-I and pWFIIP19 as a vector expressing p19 ofHTLV-II. DNA sequences of the FDX-fused HTLV-I p19 and FDX-fused HTLV-IIp19 each of which is inserted into the vectors are shown in SEQ ID NO: 6and 8, respectively, and amino acids sequences coded by said DNAsequences are shown in SEQ ID NO: 7 and 9, respectively. In the samemanner as in Example 1, these vectors were introduced into Escherichiacoli, and expression of the respective fused proteins was induced.Samples for electrophoresis were prepared under the same conditions asin Example 1. After subjecting to 12.5% SDS-PAGE according to theLaemmli method, one sheet of gel was subjected to CBB staining, and theother sheet was transferred to nitrocellulose membranes by the methodshown in Example 3. By using an anti-native HTLV-I p19 monoclonalantibody (a GIN-7 antibody, Tanaka, Y. et al., Gann., Vol.74, pp.327 to330 (1983)) or an anti-native HTLV-II p19 monoclonal antibody as aprimary antibody, and a POD-labeled antimouse IgG as a secondaryantibody, these were reacted with the fused proteins by the same methodas in Example 3 and coloring was carried out by using4-chloro-1-naphthol and hydrogen peroxide, expression of the fusedproteins reacting with the respective monoclonal antibodiescorresponding to the respective fused proteins was recognized. Thesefused proteins gave a band at about 34 Kda which was the same positionas that of the CBB-stained gels. The expression amounts of the FDX-fusedHTLV-I p19 antigen and the FDX-fused HTLV-II p19 antigen were increasedby several hundreds times as compared with the case where the p19antigen of HTLV-I and the p19 antigen of HTLV-II were expresseddirectly.

Example 6 Expression of FDX-fused HTLV-I p20E(gp21)-Fused Protein andFDX-Fused HTLV-II p20E(gp21)-Fused Protein

By the same method as in Example 5, by using DNA of cells infected withHTLV-I and HTLV-II, about 500 bp of p20E(gp21) DNA fragments in therespective env regions were obtained by the PCR method. These DNAfragments were integrated into EcoRI and BamHI sites of pWF6A preparedin Example 1 to prepare pWFIE1 as a vector expressing p20E of HTLV-I andpWFIIE10 as a vector expressing p20E of HTLV-II. DNA sequences of theFDX-fused HTLV-I p20E and FDX-fused HTLV-II p20E each of which isinserted into the vectors are shown in SEQ ID NO: 10 and 12,respectively, and amino acids sequences coded by said DNA sequences areshown in SEQ ID NO: 11 and 13, respectively. These vectors wereintroduced into Escherichia coli, and expression of a FDX-fused HTLV-Ip20E-fused protein (hereinafter referred to as “FDX-20(I)” in thespecification) and a FDX-fused HTLV-II p20E-fused protein (hereinafterreferred to as “FDX-20(II)” in the specification) was induced under thesame conditions as in Example 1. In the same manner as in Example 1,Escherichia coli was sonicated. After subjecting to 12.5% SDS-PAGEaccording to the Laemmli method, one sheet of gel was subjected to CBBstaining, and the other sheet of gel was transferred to nitrocellulosemembranes at 120 mA for 3 hours. After blocking the protein portionadsorbed to the nitrocellulose membranes with a phosphate buffercontaining 1% of BSA (bovine serum albumin), 1 μg/ml of ananti-p20E(gp21) monoclonal antibody (F-10, Sugamura, K. et al., J.Immunol., Vol.132, pp.3180 to 3184 (1984)) reacting with p20E(gp21)antigens of native HTLV-I and HTLV-II was reacted with the fusedproteins at room temperature for 1 hour, and then reacted with aPOD-labeled anti-mouse IgG at room temperature for 1 hour. Subsequently,when coloring was carried out by using 4-chloro-1-naphthol and hydrogenperoxide, expression of fused proteins reacting with the anti-p20E(gp21)monoclonal antibody corresponding to the respective fused proteins wasrecognized. These fused proteins gave a band at about 32 Kda which wasthe same position as that of the CBB-stained gels.

The expression amounts of FDX-20(I) and FDX-20(II) were increased byseveral hundreds times as compared with the case where p20E of HTLV-Iand p20E of HTLV-II were expressed directly.

Example 7 Purification of FDX-20(I)- and FDX-20(II)-Fused Proteins

pWFIE1 and pWFIIE10 prepared in Example 6 were introduced into hostEscherichia coli, respectively, and then cultured under conditions ofusing the LB medium at 37° C. By preculture, a concentration ofEscherichia coli in culture broths was made to have such turbidity thatabsorbance at a wavelength of 600 nm was about 1.0, 1 mM IPTG was addedthereto to induce expression. Three hours after IPTG was added,centrifugation was carried out to recover Escherichia coli. 200 ml of a50 mM Tris-hydrochloride buffer containing 1% Triton×100 (trade name,produced by Rohm & Haas Co.) and 2 M urea with pH 8.0 was added torecovered Escherichia coli, followed by sonication treatment under icecooling. Centrifugation was carried out to recover insoluble materials(inclusion bodies). The inclusion bodies were solubilized by using a 4 Mguanidine hydrochloride-10 mM dithiothreitol (hereinafter referred to as“DTT” in the specification) solution. The solubilized bodies werepurified by a RESOURCE RPC column (trade name, manufactured by PharmaciaBiotec Co.) equilibrated with 20% acetonitrile and 20 mM sodiumhydroxide. When the bodies were eluted by acetonitrile, purifiedFDX-20(I)- and FDX-20(II)-fused proteins were recovered at aconcentration of about 30 to 40% acetonitrile-eluted fractions,respectively.

Reference example 2 Purification of TRX-fused HTLV-I p20E-fused proteinand TRX-fused HTLV-II p20E-fused protein

In the same manner as in Example 6, p20E(gp21) in an env region ofHTLV-I or HTLV-II was introduced into the TRX-expressing vector pWT8Aprepared in Reference example 1 to prepare pWTIE1 and pWTIIE10, followedby expression. In the same manner as in Example 7, by the purificationmethod using a RESOURCE RPC column (trade name, manufactured byPharmacia Biotec Co.), a TRX-fused HTLV-I p20E-fused protein(hereinafter referred to as “TRX-20(I)” in the specification) and aTRX-fused HTLV-II p20E-fused protein (hereinafter referred to as“TRX-20(II)” in the specification) were purified.

Example 8 Reactivity Test of Fused Proteins

(1) Test by the Western Blotting Method

By using FDX-20(I) and FDX-20(II) purified in Example 7 and TRX-20(I)and TRX-20(II) purified in Reference example 2, reactivities with humanHTLV specimens in the western blotting method were compared.

In the same manner as in Example 3, the western blotting method wascarried out by using the human specimen HTLV-I/II mix panel 204 serumproduced by Boston Biomedica Co. diluted 50 times as primary antibodiesand POD-labelled anti-human IgG as a secondary antibody. FDX-20(I) andFDX-20(II), and TRX-20(I) and TRX-20(II) were reacted with the samespecimens, respectively. The results are shown in Table 2.

TABLE 2 Intensity of reaction (+,−) by western blotting Specimen No.FDX-20 (I) TRX-20 (I) FDX-20 (II) TRX-20 (II) PRP-204-01 + + + +PRP-204-02 − − − − PRP-204-03 + + + + PRP-204-04 − − + + PRP-204-05 + +− − PRP-204-06 − − − − PRP-204-07 + + + + PRP-204-08 − − − −PRP-204-09 + + − − PRP-204-10 + + + + PRP-204-11 + + + + PRP-204-12 ++++ ++ ++ PRP-204-13 + + + + PRP-204-14 − − + + PRP-204-15 + + + +PRP-204-16 − − + + PRP-204-17 + + + + PRP-204-18 + + + + PRP-204-19 + +− − PRP-204-20 − − − − PRP-204-21 + + + + PRP-204-22 + + + +PRP-204-23 + + + + PRP-204-24 + + + + PRP-204-25 + + + + +: positive,++: strongly positive, −: negative

(2) Comparison by the ELISA Method

On ELISA plates (produced by Becton Deckinson Co.) were sensitized each50 μl of FDX-20(I) and FDX-20(II) purified in Example 7 and TRX-20(I)and TRk-20(II) purified in Reference example 2 at a concentration of 3μg/ml, respectively.

The ELISA method was carried out by using these ELISA plates and usingthe human specimens produced by Boston Biomedica Co. diluted 500 timesas primary antibodies and POD-labelled anti-human IgG as a secondaryantibody in the same manner as in Example 4. FDX-20(I) and FDX-20(II),and TRX-20(I) and TRX-20(II) were reacted with the same specimens. Theresults are shown in FIG. 4 and FIG. 5.

(3) Test of Dependency on Concentration by the ELISA Method

In order to examine reactivities to the anti-p20E(gp21) monoclonalantibody and a negative serum, 10 μg/ml to ½ dilution series ofFDX-20(I) and FDX-20(II) purified in Example 7 and TRX-20(I) andTRX-20(II) purified in Reference example 2 were prepared, respectively,and ELISA plates (produced by Becton Deckinson Co.) were sensitized witheach 50 μl thereof.

The ELISA method was carried out by using these ELISA plates and usingthe anti-p20E(gp21) monoclonal antibody diluted 500 times as a primaryantibody and POD-labelled anti-mouse IgG as a secondary antibody. Withrespect to a negative serum, the ELISA method was carried out in thesame manner as in Example 4. There was no difference in reactivity tothe monoclonal antibody, and the FDX-fused proteins in both cases ofHTLV-I and HTLV-II had lower reactivities to the negative serum. Theresults are shown in FIG. 6 and FIG. 7.

Reference example 3 Preparation of protein in which GST and Treponemapallidum 15Kda antigen are fused

From syphilis bacteria (Nichols strain from Treponema pallidum) purifiedfrom pyphilis bacteria-subcultured rabbit testicles, genomic DNA wasextracted. By using the extracted DNA as a template, a primer wasproduced based on the known DNA sequences by using a DNA synthesizer(Model 392, trade name, produced by PERKIN ELMER Co.). By using theprimer, about 370 bp of a DNA fragment coding a surface antigen of 15Kda (hereinafter referred to as “Tp15” in the specification) ofTreponema pallidum (hereinafter referred to as “Tp” in thespecification) was amplified with a thermal cycler (Model PJ1000, tradename, produced by PERKIN ELMER Co.). This DNA fragment was integratedinto an EcoRI site of a GST-expressing type vector pWG6A in which DNAsequence of GST had been inserted into pW6A to obtain a vector pWGTp15expressing a protein in which GST and Tp15 were fused (hereinafterreferred to as “GST-15” in the specification). DNA sequence of theGST-15 inserted into the vector is shown in SEQ ID NO: 14 and aminoacids sequence coded by said DNA sequence is shown in SEQ ID NO: 15. Inthe same manner as in Example 1, the vector was introduced intoEscherichia coli, and expression of GST-15 was induced. A sample forelectrophoresis was prepared under the same conditions as in Example 1.After subjecting to 12.5% SDS-PAGE according to the Laemmli method, onesheet of gel was subjected to CBB staining, and the other sheet wastransferred to a nitrocellulose membrane by the method shown in Example3. By using an anti-Tp15 monoclonal antibody as a primary antibody and aPOD-labeled anti-mouse IgG as a secondary antibody, these were reactedin the same method as in Example 3 and coloring was carried out by using4-chloro-1-naphthol and hydrogen peroxide, a band was given at about 42Kda which was the same position as that of the CBB-stained gel.

Reference example 4 Preparation of protein in which TRX and Tp15 arefused

A DNA fragment of Tp15 amplified in Reference example 3 was integratedinto an EcoRI site of the TRX-expressing type vector pWT8A in which DNAsequence of TRX had been inserted into pW6A to obtain a vector pWTTp15expressing a protein in which TRX and Tp15 were fused (hereinafterreferred to as “TRX-15” in the specification). DNA sequence of theTRX-15 inserted into the vector is shown in SEQ ID NO: 16 and aminoacids sequence coded by said DNA sequence is shown in SEQ ID NO: 17. Inthe same manner as in Example 1, the vector was introduced intoEscherichia coli, and expression of TRX-15 was induced. A sample forelectro-phoresis was prepared under the same conditions as in Example 1.After subjecting to 12.5% SDS-PAGE according to the Laemmli method, onesheet of gel was subjected to CBB staining, and the other sheet wastransferred to a nitrocellulose membrane by the method shown in Example3. By using an anti-Tp15 monoclonal antibody as a primary antibody and aPOD-labeled anti-mouse IgG as a secondary anti-body, these were reactedin the same method as in Example 3 and coloring was carried out by using4-chloro-1-naphthol and hydrogen peroxide, a band was given at about 27Kda which was the same position as that of the CBB-stained gel.

Example 9 Preparation of Protein in Which FDX and Tp15 are Fused

A DNA fragment of Tp15 amplified in Reference example 3 was integratedinto an EcoRI, BamHI sites of the FDX-expressing type vector pWF6Aprepared in Example 1 to obtain a vector pWFTp15 expressing a protein inwhich FDX and Tp15 were fused (hereinafter referred to as “FDX-15” inthe specification). DNA sequence of the FDX-15 inserted into the vectoris shown in SEQ ID NO: 18 and amino acids sequence coded by said DNAsequence is shown in SEQ ID NO: 19. In the same manner as in Example 1,the vector was introduced into Escherichia coli, and expression ofFDX-15 was induced. A sample for electrophoresis was prepared under thesame conditions as in Example 1. After subjecting to 12.5% SDS-PAGEaccording to the Laemmli method, one sheet of gel was subjected to CBBstaining, and the other sheet was transferred to a nitrocellulosemembrane by the method shown in Example 3. By using an anti-Tp15monoclonal anti-body as a primary antibody and a POD-labeled anti-mouseIgG as a secondary antibody, these were reacted in the same method as inExample 3 and coloring was carried out by using 4-chloro-1-naphthol andhydrogen peroxide, a band was given at about 30 Kda which was the sameposition as that of the CBB-stained gel.

Example 10 Heat Resistance Test of FDX-15, GST-15 and TRX-15

The vectors expressing FDX-15, GST-15 and TRX-15 prepared in Example 9,Reference example 3 and Reference example 4 were introduced into hostEscherichia coli and then cultured under conditions of using 1 liter ofthe LB medium at 37° C., respectively. By preculture, a concentration ofEscherichia coli in culture broths was made to have such turbidity thatabsorbance at a wavelength of 600 nm was about 1.0, 1 mM IPTG was addedthereto to induce expression. After the cells were recovered bycentrifugation, 200 ml of the Tris buffer was added to the cells. Aftersonication treatment under ice cooling, fused proteins were recovered inthe centrifugation supernatants, respectively. 800 μl of these proteinswere taken, respectively, and shaken for 13 minutes in water bath at 40°C., 50° C., 60° C., 70° C. and 80° C. The respective samples werecentrifuged and then separated into supernatants and precipitates, andanalysis was carried out by SDS-PAGE and the western blotting method. Asa blocking agent of the western blotting method, 1% skim milk dissolvedin PBS was used, and as a primary antibody, an anti-TP rabbit antibodywas used. As a secondary antibody, a POD-labelled anti-rabbit antibodywas used, and as a coloring agent, 4-chloro-1-naphthol and hydrogenperoxide were used. The result of coloring of western blotting wasconfirmed by a densitometer. The results are shown in FIG. 8 and FIG. 9.Precipitates of TRX-15 and GST-15 were partially generated at 40° C. bythermal denaturation, about 80% of TRX-15 and GST-15 were precipitatedat 60° C., and about 100% of them were precipitated at 70° C. Almost noprecipitate by thermal denaturation of FDX-15 was generated at 40° C. to80° C., and even at 80° C., about 100% of FDX-15 existed in thesupernatant.

Example 11 Purification of FDX-15 by Heat Treatment

pWFTp15 prepared in Example 9 was introduced into host Escherichia coliand then cultured under conditions of using 1 liter of the LB medium at37° C. By preculture, a concentration of Escherichia coli in culturebroths was made to have such turbidity that absorbance at a wavelengthof 600 nm was about 1.0, 1 mM IPTG was added thereto to induceexpression. The cells were recovered by centrifugation. 200 ml of theTris buffer was added to the cells, and the cells were sonicated torecover FDX-15 in the centrifugation supernatant. Then, by using a hotplate and a water bath, heat treatment at 70° C. for 10 minutes wascarried out to recover FDX-15 in the centrifugation supernatant. Thesupernatant subjected to heat treatment was purified by a QFF anionexchange column (trade name, manufactured by Pharmacia Biotec Co.)equilibrated with the Tris buffer. When the supernatant was eluted by acolumn equilibrated buffer containing sodium chloride, FDX-15 wasrecovered at a concentration of about 0.3 M to 0.4 M sodiumchloride-eluted fraction. Then, 10 mM DTT was added to the QFF recoveredfraction, and the mixture was purified by using a RESOURCE RPC column(trade name, manufactured by Pharmacia Biotec Co.) equilibrated with a20 mM sodium hydroxide solution. When the mixture was eluted byacetonitrile, FDX-15 was recovered at a concentration of about 20% to25% acetonitrile-eluted fraction. This reverse phase recovered fractionwas concentrated by Centriprep (trade name, manufactured by AmiconInc.), and the concentrate was subjected to gel filtration by a Superdex200 column (trade name, manufactured by Pharmacia Biotec Co.). When thefiltrate was eluted by a buffer containing 6 M urea, 0.5 M sodiumchloride and 20 mM Tris-hydrochloride having pH 8.0, purified FDX-15 wasrecovered at a molecular weight of about 50,000. By heat treatment at60° C., about 80% of the Escherichia coli protein was precipitated bythermal denaturation, but even at 70° C., almost 100% of FDX-15 wasrecovered in the supernatant, and the purification degree was raised byabout 5 times only by heat treatment.

Further, GST-15 obtained by introducing pWGTp15 prepared in Referenceexample 3 into host Escherichia coli, carrying out induction andexpression operations in the same manner therein and carrying outpurification by a common column operation without carrying out heattreatment and FDX-15 purified by heat treatment were subjected to thewestern blotting method in the same manner as in Example 10 by using ananti-Tp rabbit antibody. It was shown that even though purification byheat treatment was carried out, FDX-15 retained reactivity. The resultsare shown in FIG. 10.

Example 12 Preparation of AK-expressing Vector pW6AK

By using 16 primers of 53 mer prepared based on a known DNA sequence ofAK derived from a Sulfolobus bacterium by using a DNA synthesizer(manufactured by Perkin Elmer Co.), genes of Sulfolobus acidocaldariusAK were synthesized by the assemble PCR method. In the assemble PCRmethod, a Taq polymerase (produced by Toyobo Co.) was used, and thetotal base number of 630 bp was amplified under conditions of 30 cyclesof 94° C.−1 minute, 55° C.−1 minute and 72° C.−1 minute. A restrictionenzyme NdeI site was added to 5′-end, a restriction enzyme EcoRI sitewas added to 3′-end, and a thrombin-cut site was added to C terminal.This fragment was integrated into the NdeI and EcoRI sites of 4.6 Kb ofa pW6A vector prepared from pGEMEX-1 (trade name, produced by PromegaCo.) and pGEX-2T (trade name, produced by Pharmacia Biotec Co.) toprepare pW6AK as a vector expressing AK. A detailed view of pW6AK isshown in FIG. 11. pW6AK contains genes of a fused protein comprising 223amino acids including 194 amino acids derived from AK, 10 amino acidsderived from a thrombin-cleaved site and 19 amino acids derived frommulti cloning site of pW6A, at the NdeI and EcoRI sites. The basesequence of the inserted fragment was confirmed by a DNA sequence kit(trade name: Sequenase kit Ver. 2.0, produced by Amersham United StatesBiochemical Co.). DNA sequence of the AK inserted into the pW6A is shownin SEQ ID NO: 3 and amino acids sequence coded by said DNA sequence isshown in SEQ ID NO: 4. pW6AK was introduced into host Escherichia coliand then cultured for 2 hours in the LB medium. Thereafter, 1 mM IPTGwas added thereto, and the mixture was cultured for 2 hours to induceexpression. The TE buffer were added to the precipitates of Escherichiacoli, the precipitates were sonicated, and 15% SDS-PAGE according to theLaemmli method was carried out. By CBB staining, a band was confirmed atabout 40 Kda.

Example 13 Purification of AK

pW6AK prepared in Example 12 was introduced into host

Escherichia coli and then cultured under conditions of using the LBmedium at 37° C. By preculture, a concentration of Escherichia coli inculture broth was made to have such turbidity that absorbance at awavelength of 600 nm was about 1.0, 1 mM IPTG was added thereto toinduce expression. After the mixture was cultured for 3 hours,centrifugation was carried out to recover Escherichia coli. 200 ml ofthe Tris buffer was added to recover Escherichia coli, followed bysonication treatment under ice cooling. After centrifugation, theexpressed fused protein was recovered in the supernatant as a solublecomponent. When this supernatant was subjected to heat treatment at 65°C. for 10 minutes, about 70% of the Escherichia coli protein wasthermally denatured and precipitated, and 80% or more of AK wasrecovered in the centrifugation supernatant after the heat treatment.

This supernatant was purified by a Hydroxy apatite column (manufacturedby Bio-rad Lab.) equilibrated with the Tris buffer. When the supernatantwas eluted by a sodium phosphate buffer, AK was recovered at aconcentration of about 0.2 M sodium phosphate-eluted fraction. Then,this AK fraction was purified by gel filtration using a Superdex 20026/60 column (trade name, manufactured by Pharmacia Biotec Co.)equilibrated with a buffer containing 6 M urea, 0.5 M sodium chlorideand 20 mM Tris-hydrochloride having pH 9.4. At a fraction of a molecularweight being about 20,000, purified AK was recovered.

Example 14 Preparation of Protein in Which AK and Tp15 are Fused

A DNA fragment of Tp15 amplified in Reference example 3 was integratedinto EcoRI BamHI sites of the AK-expressing type vector pW6AK preparedin Example 12 to obtain a vector pW6AKTp15 expressing a protein in whichAK and Tp15 were fused (hereinafter referred to as “AK-15” in thespecification). DNA sequence of the AK-15 inserted into the vector isshown in SEQ ID NO: 20 and amino acids sequence coded by said DNAsequence is shown in SEQ ID NO: 21. In the same manner as in Example 1,the vector was introduced into Escherichia coli, and expression of AK-15was induced. A sample for electro-phoresis was prepared under the sameconditions as in Example 1. After subjecting to 12.5% SDS-PAGE accordingto the Laemmli method, one sheet of gel was subjected to CBB staining,and the other sheet was transferred to a nitrocellulose membrane by themethod shown in Example 3. By using an anti-Tp15 monocional antibody asa primary antibody and a POD-labeled anti-mouse IgG as a secondaryanti-body, these were reacted in the same method as in Example 3 andcoloring was carried out by using 4-chloro-1-naphthol and hydrogenperoxide, a band was given at about 40 Kda which was the same positionas that of the CBB-stained gel.

Example 15 Purification of AK-15 by Heat Treatment

pWAKTp15 prepared in Example 14 was introduced into host Escherichiacoli and then cultured under conditions of using 1 liter of the LBmedium at 37° C. By preculture, a concentration of Escherichia coli inculture broth was made to have such turbidity that absorbance at awavelength of 600 nm was about 1.0, 1 mM IPTG was added thereto toinduce expression. The cells were recovered by centrifugation. 200 ml ofa 50 mM glycine-sodium hydroxide buffer having pH 10.0 was added to thecells, and the cells were sonicated to recover AK-15 in thecentrifugation supernatant. Then, by using a hot plate, heat treatmentat 60° C. for 10 minutes was carried out to recover AK-15 in thecentrifugation supernatant. The supernatant subjected to heat treatmentwas dialyzed to a 4 M urea-50 mM sodium acetate buffer having pH 6.0 andthen purified by a SFF cation exchange column (trade name, manufacturedby Pharmacia Biotec Co.) equilibrated with said buffer. When thesupernatant was eluted by a column equilibrated buffer containing sodiumchloride, AK-15 was recovered at a concentration of about 0.2 M to 0.4 Msodium chloride-eluted fraction. The recovered AK-15 fraction waspurified by gel filtration using a Superdex 200 26/60 column (tradename, manufactured by Pharmacia Biotec Co.) equilibrated with a buffercontaining 6 M urea, 0.5 M sodium chloride and 20 mM Tris-hydrochloridehaving pH 9.4. At a fraction of a molecular weight being about 40,000,purified AK-15 was recovered.

When the western blotting method was carried out in the same manner asin Example 1 by using an anti-Tp rabbit antibody, it was shown that eventhough purification by heat treatment was carried out, AK-15 retainedreactivity. The results are shown in FIG. 12.

According to the present invention; a fused DNA sequence having moreexcellent operatability and productivity than those of a conventionalDNA sequence coding a fused protein, a fused protein expressed from saidfused DNA sequence, and a method for expressing the fused protein byusing said DNA sequence are provided.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 21(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 291 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (vi)ORIGINAL SOURCE: (A) ORGANISM: SYNTHESIZED (x) PUBLICATION INFORMATION:(A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSEDPROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD FOR EXPRESSINGSAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 291(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGGCGTGGA AGGTTTCTGT CGACCAAGACACCTGTATAG GAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAG ACGTCTTTGA GATGAACGATGAAGGAAAGG CCCAACCAAA GGTAGAGGTT 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCTAAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCTTCTCTGGTTC CGCGTGGATC GGAATTCGTC 240 GACCTCGAGG GATCCGGGCC CTCTAGATGCGGCCGCATGC ATGGTACCTA A 291 (2) INFORMATION FOR SEQ ID NO:2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 96 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (vi) ORIGINAL SOURCE: (A) ORGANISM: RECOMBINANT (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:2: FROM 1 TO 96 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala TrpLys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp Ala 1 5 10 15 Ile CysAla Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp Glu Gly 20 25 30 Lys AlaGln Pro Lys Val Glu Val Ile Glu Asp Glu Glu Leu Tyr Asn 35 40 45 Cys AlaLys Glu Ala Met Glu Ala Cys Pro Val Ser Ala Ile Thr Ile 50 55 60 Glu GluAla Gly Gly Ser Ser Leu Val Pro Arg Gly Ser Glu Phe Val 65 70 75 80 AspLeu Glu Gly Ser Gly Pro Ser Arg Cys Gly Arg Met His Gly Thr 85 90 95 (2)INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:672 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (vi) ORIGINALSOURCE: (A) ORGANISM: SYNTHESIZED (x) PUBLICATION INFORMATION: (A)AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSEDPROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSINGSAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:3: FROM 1 TO 672(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGAAGATTG GTATTGTAAC TGGTATCCCTGGTGTAGGGA AAAGTACTGT CTTGGCTAAA 60 GTTAAAGAGA TATTGGATAA TCAAGGTATAAATAACAAGA TCATAAATTA TGGAGATTTT 120 ATGTTAGCAA CAGCATTAAA ATTAGGCTATGCTAAAGATA GAGACGAAAT GAGAAAATTA 180 TCTGTAGAAA AGCAGAAGAA ATTGCAGATTGATGCGGCTA AAGGTATAGC TGAAGAGGCA 240 AGAGCAGGTG GAGAAGGATA TCTGTTCATAGATACGCACG CTGTGATACG TACACCCTCT 300 GGATATTTAC CTGGTTTACC GTCAGATATAATTACAGAAA TAAATCCGTC TGTTATCTTT 360 TTACTGGAAG CTGATCCTAA GATAATATTATCAAGGCAAA AGAGAGATAC AACAAGGAAT 420 AGAAATGATT ATAGTGACGA ATCAGTTATATTAGAAACCA TAAACTTCGC TAGATATGCA 480 GCTACTGCTT CTGCAGTATT AGCCGGTTCTACTGTTAAGG TAATTGTAAA CGTGGAAGGA 540 GATCCTAGTA TAGCAGCTAA TGAGATAATAAGGTCTATGA AGGGTGGTTC TTCTCTGGTT 600 CCGCGTGGAC TGGAATTCGT CGACCTCGAGGGATCCGGGC CCTCTAGATG CGGCCGCATG 660 CATGGTACCT AA 672 (2) INFORMATIONFOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 223 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:RECOMBINANT (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJII ETAL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAIDFUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:4: FROM 1 TO 223 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:4: Met Lys Ile Gly Ile Val Thr Gly Ile Pro GlyVal Gly Lys Ser Thr 1 5 10 15 Val Leu Ala Lys Val Lys Glu Ile Leu AspAsn Gln Gly Ile Asn Asn 20 25 30 Lys Ile Ile Asn Tyr Gly Asp Phe Met LeuAla Thr Ala Leu Lys Leu 35 40 45 Gly Tyr Ala Lys Asp Arg Asp Glu Met ArgLys Leu Ser Val Glu Lys 50 55 60 Gln Lys Lys Leu Gln Ile Asp Ala Ala LysGly Ile Ala Glu Glu Ala 65 70 75 80 Arg Ala Gly Gly Glu Gly Tyr Leu PheIle Asp Thr His Ala Val Ile 85 90 95 Arg Thr Pro Ser Gly Tyr Leu Pro GlyLeu Pro Ser Asp Ile Ile Thr 100 105 110 Glu Ile Asn Pro Ser Val Ile PheLeu Leu Glu Ala Asp Pro Lys Ile 115 120 125 Ile Leu Ser Arg Gln Lys ArgAsp Thr Thr Arg Asn Arg Asn Asp Tyr 130 135 140 Ser Asp Glu Ser Val IleLeu Glu Thr Ile Asn Phe Ala Arg Tyr Ala 145 150 155 160 Ala Thr Ala SerAla Val Leu Ala Gly Ser Thr Val Lys Val Ile Val 165 170 175 Asn Val GluGly Asp Pro Ser Ile Ala Ala Asn Glu Ile Ile Arg Ser 180 185 190 Met LysGly Gly Ser Ser Leu Val Pro Arg Gly Leu Glu Phe Val Asp 195 200 205 LeuGlu Gly Ser Gly Pro Ser Arg Cys Gly Arg Met His Gly Thr 210 215 220 (2)INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:4557 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: circular (ii) MOLECULE TYPE: other nucleic acid (vi) ORIGINALSOURCE: (A) ORGANISM: E. COLI (B) STRAIN: BL21 (DE3) (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:5: FROM 1 TO 4557 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGGCTAGCGAATTCGTCGA CCTCGAGGGA TCCGGGCCCT CTAGATGCGG CCGCATGCAT 60 GGTACCTAACTAACTAAGCT TGAGTATTCT ATAGTGTCAC CTAAATCCCA GCTTGATCCG 120 GCTGCTAACAAAGCCCGAAA GGAAGCTGAG TTGGCTGCTG CCACCGCTGA GCAATAACTA 180 GCATAACCCCTTGGGGCCTC TAAACGGGTC TTGAGGGGTT TTTTGCTGAA AGGAGGAACT 240 ATATCCGGATAACCTGGCGT AATAGCGAAG AGGCCCGCAC CGAATTAATT CATCGTGACT 300 GACTGACGATCTGCCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC 360 TCCCGGAGACGGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG 420 GCGCGTCAGCGGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCCAGTCA CGTAGCGATA 480 GCGGAGTGTATAATTCTTGA AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 540 ATGTCATGATAATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG 600 GAACCCCTATTTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT 660 AACCCTGATAAATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 720 GTGTCGCCCTTATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA 780 CGCTGGTGAAAGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 840 TGGATCTCAACAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA 900 TGAGCACTTTTAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTGTTGAC GCCGGGCAAG 960 AGCAACTCGGTCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 1020 CAGAAAAGCATCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA 1080 TGAGTGATAACACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 1140 CCGCTTTTTTGCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 1200 TGAATGAAGCCATACCAAAC GACGAGCGTG ACACCACGAT GCCTGCAGCA ATGGCAACAA 1260 CGTTGCGCAAACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 1320 ACTGGATGGAGGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 1380 GGTTTATTGCTGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 1440 TGGGGCCAGATGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA 1500 CTATGGATGAACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT 1560 AACTGTCAGACCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 1620 TTAAAAGGATCTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 1680 AGTTTTCGTTCCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 1740 CTTTTTTTCTGCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 1800 TTTGTTTGCCGGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 1860 CGCAGATACCAAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 1920 CTGTAGCACCGCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 1980 GCGATAAGTCGTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 2040 GGTCGGGCTGAACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 2100 AACTGAGATACCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 2160 CGGACAGGTATCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 2220 GGGGAAACGCCTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC 2280 GATTTTTGTGATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 2340 TTTTACGGTTCCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 2400 CTGATTCTGTGGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC 2460 GAACGACCGAGCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCTG ATGCGGTATT 2520 TTCTCCTTACGCATCTGTGC GGTATTTCAC ACCGCATAAA TTCCGACACC ATCGAATGGT 2580 GCAAAACCTTTCGCGGTATG GCATGATAGC GCCCGGAAGA GAGTCAATTC AGGGTGGTGA 2640 ATGTGAAACCAGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG 2700 TTTCCCGCGTGGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG 2760 CGGCGATGGCGGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG GCGGGCAAAC 2820 AGTCGTTGCTGATTGGCGTT GCCACCTCCA GTCTGGCCCT GCACGCGCCG TCGCAAATTG 2880 TCGCGGCGATTAAATCTCGC GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG 2940 AACGAAGCGGCGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA 3000 GTGGGCTGATCATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT 3060 GCACTAATGTTCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC AACAGTATTA 3120 TTTTCTCCCATGAAGACGGT ACGCGACTGG GCGTGGAGCA TCTGGTCGCA TTGGGTCACC 3180 AGCAAATCGCGCTGTTAGCG GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG 3240 GCTGGCATAAATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT 3300 GGAGTGCCATGTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA 3360 CTGCGATGCTGGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC ATTACCGAGT 3420 CCGGGCTGCGCGTTGGTGCG GATATCTCGG TAGTGGGATA CGACGATACC GAAGACAGCT 3480 CATGTTATATCCCGCCGTTA ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA 3540 GCGTGGACCGCTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC 3600 CCGTCTCACTGGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC 3660 GCGCGTTGGCCGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC 3720 AGTGAGCGCAACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC 3780 TTTATGCTTCCGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT TCACACAGGA 3840 AACAGCTATGACCATGATTA CGGATTCACT GGCCGTCGTT TTACAACGTC GTGACTGGGA 3900 AAACCCTGGCGTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTTTCG CCAGCTGGCG 3960 TAATAGCGAAGAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA 4020 ATGGCGCTTTGCCTGGTTTC CGGCACCAGA AGCGGTGCCG GAAAGCTGGC TGGAGTGCGA 4080 TCTTCCTGAGGCCGATACTG TCGTCGTCCC CTCAAACTGG CAGATGCACG GTTACGATGC 4140 GCCCATCTACACCAACGTAA CCTATCCCAT TACGGTCAAT CCGCCGTTTG TTCCCACGGA 4200 GAATCCGACGGGTTGTTACT CGCTCACATT TAATGTTGAT GAAAGCTGGC TACAGGAAGG 4260 CCAGACGCGAATTATTTTTG ATGGCGTTGG AATTACGTTA TCGACTGCAC GGTGCACCAA 4320 TGCTTCTGGCGTCAGGCAGC CATCGGAAGC TGTGGTATGG CTGTGCAGGT CGTAAATCAC 4380 TGCATAATTCGTGTCGCTCA AGGCGCACTC CCGTTCTGGA TAATGTTTTT TGCGCCGACA 4440 TCATAACGGTTCTGGCAAAT GGGAATTGGG AAATTAATAC GACTCACTAT ATGGAATTGT 4500 GAGCGGATAACAATTCCTAG AAATAATTTT GTTTAACTTT AAGAAGGAGA TATACAT 4557 (2) INFORMATIONFOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 672 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A)ORGANISM: SYNTHESIZED, HTLV-1 (x) PUBLICATION INFORMATION: (A) AUTHORS:NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEINEXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAIDFUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:6: FROM 1 TO 672 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGGCGTGGA AGGTTTCTGT CGACCAAGACACCTGTATAG GAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAG ACGTCTTTGA GATGAACGATGAAGGAAAGG CCCAACCAAA GGTAGAGGTT 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCTAAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCTTCTCTGGTTC CGCGTGGATC GGAATTCATG 240 GGCCAAATCT TTTCCCGTAG CGCTAGCCCTATTCCGCGGC CGCCCCGGGG GCTGGCCGCT 300 CATCACTGGC TTAACTTCCT CCAGGCGGCATATCGCCTAG AACCCGGTCC CTCCAGTTAC 360 GATTTCCACC AGTTAAAAAA ATTTCTTAAAATAGCTTTAG AAACACCGGT CTGGATCTGC 420 CCCATTAACT ACTCCCTCCT AGCCAGCCTACTCCCAAAAG GATACCCCGG CCGGGTGAAT 480 GAAATTTTAC ACATACTCAT CCAAACCCAAGCCCAGATCC CGTCCCGCCC CGCGCCGCCG 540 CCGCCGTCAT CCTCCACCCA CGACCCCCCGGATTCTGACC CACAAATCCC CCCTCCCTAT 600 GTTGAGCCTA CAGCCCCCCA AGTCCTTTAAGGATCCGGGC CCTCTAGATG CGGCCGCATG 660 CATGGTACCT AA 672 (2) INFORMATIONFOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 209 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:RECOMBINANT (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJII ETAL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAIDFUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:7: FROM 1 TO 209 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:7: Met Ala Trp Lys Val Ser Val Asp Gln Asp ThrCys Ile Gly Asp Ala 1 5 10 15 Ile Cys Ala Ser Leu Cys Pro Asp Val PheGlu Met Asn Asp Glu Gly 20 25 30 Lys Ala Gln Pro Lys Val Glu Val Ile GluAsp Glu Glu Leu Tyr Asn 35 40 45 Cys Ala Lys Glu Ala Met Glu Ala Cys ProVal Ser Ala Ile Thr Ile 50 55 60 Glu Glu Ala Gly Gly Ser Ser Leu Val ProArg Gly Ser Glu Phe Met 65 70 75 80 Gly Gln Ile Phe Ser Arg Ser Ala SerPro Ile Pro Arg Pro Pro Arg 85 90 95 Gly Leu Ala Ala His His Trp Leu AsnPhe Leu Gln Ala Ala Tyr Arg 100 105 110 Leu Glu Pro Gly Pro Ser Ser TyrAsp Phe His Gln Leu Lys Lys Phe 115 120 125 Leu Lys Ile Ala Leu Glu ThrPro Val Trp Ile Cys Pro Ile Asn Tyr 130 135 140 Ser Leu Leu Ala Ser LeuLeu Pro Lys Gly Tyr Pro Gly Arg Val Asn 145 150 155 160 Glu Ile Leu HisIle Leu Ile Gln Thr Gln Ala Gln Ile Pro Ser Arg 165 170 175 Pro Ala ProPro Pro Pro Ser Ser Ser Thr His Asp Pro Pro Asp Ser 180 185 190 Asp ProGln Ile Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gln Val 195 200 205 Leu(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 690 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINALSOURCE: (A) ORGANISM: SYNTHESIZED, HTLV-II (x) PUBLICATION INFORMATION:(A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSEDPROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSINGSAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:8: FROM 1 TO 690(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATGGCGTGGA AGGTTTCTGT CGACCAAGACACCTGTATAG GAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAG ACGTCTTTGA GATGAACGATGAAGGAAAGG CCCAACCAAA GGTAGAGGTT 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCTAAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCTTCTCTGGTTC CGCGTGGATC GGAATTCATG 240 GGACAAATCC ACGGGCTTTC CCCAACTCCAATACCCAAAG CCCCCAGGGG GCTATCAACC 300 CACCACTGGC TTAACTTTCT CCAGGCTGCTTACCGCTTGC AGCCTAGGCC CTCCGATTTC 360 GACTTCCAGC AGCTACGACG CTTTCTAAAACTAGCCCTTA AAACGCCCAT TTGGCTAAAT 420 CCTATTGACT ACTCGCTTTT AGCTAGCCTTATCCCCAAGG GATATCCAGG AAGGGTGGTA 480 GAGATTATAA ATATCCTTGT CAAAAATCAAGTCTCCCCTA GCGCCCCCGC CGCCCCAGTT 540 CCGACACCTA TCTGCCCTAC TACTACTCCTCCGCCACCTC CCCCCCCTTC CCCGGAGGCC 600 CATGTTCCCC CCCCTTACGT GGAACCCACCACCACGCAAT GCTTCTAAGG ATCCGGGCCC 660 TCTAGATGCG GCCGCATGCA TGGTACCTAA690 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 215 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:(A) ORGANISM: RECOMBINANT (x) PUBLICATION INFORMATION: (A) AUTHORS:NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEINEXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAIDFUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:9: FROM 1 TO 215 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Trp Lys Val Ser Val Asp GlnAsp Thr Cys Ile Gly Asp Ala 1 5 10 15 Ile Cys Ala Ser Leu Cys Pro AspVal Phe Glu Met Asn Asp Glu Gly 20 25 30 Lys Ala Gln Pro Lys Val Glu ValIle Glu Asp Glu Glu Leu Tyr Asn 35 40 45 Cys Ala Lys Glu Ala Met Glu AlaCys Pro Val Ser Ala Ile Thr Ile 50 55 60 Glu Glu Ala Gly Gly Ser Ser LeuVal Pro Arg Gly Ser Glu Phe Met 65 70 75 80 Gly Gln Ile His Gly Leu SerPro Thr Pro Ile Pro Lys Ala Pro Arg 85 90 95 Gly Leu Ser Thr His His TrpLeu Asn Phe Leu Gln Ala Ala Tyr Arg 100 105 110 Leu Gln Pro Arg Pro SerAsp Phe Asp Phe Gln Gln Leu Arg Arg Phe 115 120 125 Leu Lys Leu Ala LeuLys Thr Pro Ile Trp Leu Asn Pro Ile Asp Tyr 130 135 140 Ser Leu Leu AlaSer Leu Ile Pro Lys Gly Tyr Pro Gly Arg Val Val 145 150 155 160 Glu IleIle Asn Ile Leu Val Lys Asn Gln Val Ser Pro Ser Ala Pro 165 170 175 AlaAla Pro Val Pro Thr Pro Ile Cys Pro Thr Thr Thr Pro Pro Pro 180 185 190Pro Pro Pro Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr Val Glu 195 200205 Pro Thr Thr Thr Gln Cys Phe 210 215 (2) INFORMATION FOR SEQ IDNO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 810 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM:SYNTHESIZED, HTLV-I (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKIFUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROMSAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:10: FROM 1 TO 810 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:10: ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAGGAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGGCCCAACCAAA GGTAGAGGTT 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTATGGAGGCCTG TCCAGTTAGT 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTCCGCGTGGATC GGAATTCGCA 240 GTACCGGTGG CGGTCTGGCT TGTCTCCGCC CTGGCCATGGGAGCCGGAGT GGCTGGCAGG 300 ATTACCGGCT CCATGTCCCT CGCCTCAGGA AAGAGCCTCCTACATGAGGT GGACAAAGAT 360 ATTTCCCAAT TAACTCAAGC AATAGTCAAA AACCACAAAAATCTGCTCAA AATTGCACAG 420 TATGCTGCCC AGAACAGACG AGGCCTTGAT CTCCTGTTCTGGGAGCAAGG AGGATTATGC 480 AAAGCATTAC AAGAACAGTG CTGTTTTCTA AATATTACTAATTCCCATGT CTCAATACTA 540 CAAGAGAGAC CCCCCCTTGA AAATCGAGTC CTGACTGGCTGGGGCCTTAA CTGGGACCTT 600 GGCCTCTCAC AGTGGGCTCG AGAAGCCTTA CAAACTGGAATCACCCTTGT CGCGCTACTC 660 CTTCTTGTTA TCCTTGCAGG ACCATGCATC CTCCGTCAGCTACGACACCT CCCCTCGCGC 720 GTCAGATACC CCCATTACTC TCTTATAAAC CCTGAGTCATCCCTGTAAGG ATCCGGGCCC 780 TCTAGATGCG GCCGCATGCA TGGTACCTAA 810 (2)INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:255 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A)ORGANISM: RECOMBINANT (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKIFUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROMSAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:11: FROM 1 TO 255 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:11: Met Ala Trp Lys Val Ser Val Asp Gln Asp ThrCys Ile Gly Asp Ala 1 5 10 15 Ile Cys Ala Ser Leu Cys Pro Asp Val PheGlu Met Asn Asp Glu Gly 20 25 30 Lys Ala Gln Pro Lys Val Glu Val Ile GluAsp Glu Glu Leu Tyr Asn 35 40 45 Cys Ala Lys Glu Ala Met Glu Ala Cys ProVal Ser Ala Ile Thr Ile 50 55 60 Glu Glu Ala Gly Gly Ser Ser Leu Val ProArg Gly Ser Glu Phe Ala 65 70 75 80 Val Pro Val Ala Val Trp Leu Val SerAla Leu Ala Met Gly Ala Gly 85 90 95 Val Ala Gly Arg Ile Thr Gly Ser MetSer Leu Ala Ser Gly Lys Ser 100 105 110 Leu Leu His Glu Val Asp Lys AspIle Ser Gln Leu Thr Gln Ala Ile 115 120 125 Val Lys Asn His Lys Asn LeuLeu Lys Ile Ala Gln Tyr Ala Ala Gln 130 135 140 Asn Arg Arg Gly Leu AspLeu Leu Phe Trp Glu Gln Gly Gly Leu Cys 145 150 155 160 Lys Ala Leu GlnGlu Gln Cys Cys Phe Leu Asn Ile Thr Asn Ser His 165 170 175 Val Ser IleLeu Gln Glu Arg Pro Pro Leu Glu Asn Arg Val Leu Thr 180 185 190 Gly TrpGly Leu Asn Trp Asp Leu Gly Leu Ser Gln Trp Ala Arg Glu 195 200 205 AlaLeu Gln Thr Gly Ile Thr Leu Val Ala Leu Leu Leu Leu Val Ile 210 215 220Leu Ala Gly Pro Cys Ile Leu Arg Gln Leu Arg His Leu Pro Ser Arg 225 230235 240 Val Arg Tyr Pro His Tyr Ser Leu Ile Asn Pro Glu Ser Ser Leu 245250 255 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 816 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi)ORIGINAL SOURCE: (A) ORGANISM: SYNTHESIZED, HTLV-II (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:12: FROM 1 TO 816 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATGGCGTGGAAGGTTTCTGT CGACCAAGAC ACCTGTATAG GAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAGACGTCTTTGA GATGAACGAT GAAGGAAAGG CCCAACCAAA GGTAGAGGTT 120 ATTGAGGACGAAGAGCTCTA CAACTGTGCT AAGGAAGCTA TGGAGGCCTG TCCAGTTAGT 180 GCTATTACTATTGAGGAGGC TGGTGGTTCT TCTCTGGTTC CGCGTGGATC GGAATTCGCC 240 GTTCCAATAGCAGTGTGGCT TGTCTCCGCC CTAGCGGCCG GAACAGGTAT CGCTGGTGGA 300 GTAACAGGCTCCCTATCTCT GGCTTCCAGT AAAAGCCTTC TCCTCGAGGT TGACAAAGAC 360 ATCTCCCACCTTACCCAGGC CATAGTCAAA AATCATCAAA ACATCCTCCG GGTTGCACAG 420 TATGCAGCCCAAAATAGACG AGGATTAGAC CTCCTATTCT GGGAACAAGG GGGTTTGTGC 480 AAGGCCATACAGGAGCAATG TTGCTTCCTC AACATCAGTA ACACTCATGT ATCCGTCCTC 540 CAGGAACGGCCCCCTCTTGA AAAACGTGTC ATCACCGGCT GGGGACTAAA CTGGGATCTT 600 GGACTGTCCCAATGGGCACG AGAAGCCCTC CAGACAGGCA TAACCATTCT CGCTCTACTC 660 CTCCTCGTCATATTGTTTGG CCCCTGTATC CTCCGCCAAA TCCAGGCCCT TCCACAGCGG 720 TTACAAAACCGACATAACCA GTATTCCCTT ATCAACCCAG AAACCATGCT ATAAGGATCC 780 GGGCCCTCTAGATGCGGCCG CATGCATGGT ACCTAA 816 (2) INFORMATION FOR SEQ ID NO:13: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 257 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (vi) ORIGINAL SOURCE: (A) ORGANISM: RECOMBINANT (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:13: FROM 1 TO 257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met AlaTrp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly Asp Ala 1 5 10 15 IleCys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn Asp Glu Gly 20 25 30 LysAla Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu Leu Tyr Asn 35 40 45 CysAla Lys Glu Ala Met Glu Ala Cys Pro Val Ser Ala Ile Thr Ile 50 55 60 GluGlu Ala Gly Gly Ser Ser Leu Val Pro Arg Gly Ser Glu Phe Ala 65 70 75 80Val Pro Ile Ala Val Trp Leu Val Ser Ala Leu Ala Ala Gly Thr Gly 85 90 95Ile Ala Gly Gly Val Thr Gly Ser Leu Ser Leu Ala Ser Ser Lys Ser 100 105110 Leu Leu Leu Glu Val Asp Lys Asp Ile Ser His Leu Thr Gln Ala Ile 115120 125 Val Lys Asn His Gln Asn Ile Leu Arg Val Ala Gln Tyr Ala Ala Gln130 135 140 Asn Arg Arg Gly Leu Asp Leu Leu Phe Trp Glu Gln Gly Gly LeuCys 145 150 155 160 Lys Ala Ile Gln Glu Gln Cys Cys Phe Leu Asn Ile SerAsn Thr His 165 170 175 Val Ser Val Leu Gln Glu Arg Pro Pro Leu Glu LysArg Val Ile Thr 180 185 190 Gly Trp Gly Leu Asn Trp Asp Leu Gly Leu SerGln Trp Ala Arg Glu 195 200 205 Ala Leu Gln Thr Gly Ile Thr Ile Leu AlaLeu Leu Leu Leu Val Ile 210 215 220 Leu Phe Gly Pro Cys Ile Leu Arg GlnIle Gln Ala Leu Pro Gln Arg 225 230 235 240 Leu Gln Asn Arg His Asn GlnTyr Ser Leu Ile Asn Pro Glu Thr Met 245 250 255 Leu (2) INFORMATION FORSEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1119 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM:PLASMID, Tp (B) STRAIN: NICHOLS (x) PUBLICATION INFORMATION: (A)AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSEDPROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSINGSAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ ID NO:14: FROM 1 TO 1119(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATGTCCCCTA TACTAGGTTATTGGAAAATT AAGGGCCTTG TGCAACCCAC TCGACTTCTT 60 TTGGAATATC TTGAAGAAAAATATGAAGAG CATTTGTATG AGCGCGATGA AGGTGATAAA 120 TGGCGAAACA AAAAGTTTGAATTGGGTTTG GAGTTTCCCA ATCTTCCTTA TTATATTGAT 180 GGTGATGTTA AATTAACACAGTCTATGGCC ATCATACGTT ATATAGCTGA CAAGCACAAC 240 ATGTTGGGTG GTTGTCCAAAAGAGCGTGCA GAGATTTCAA TGCTTGAAGG AGCGGTTTTG 300 GATATTAGAT ACGGTGTTTCGAGAATTGCA TATAGTAAAG ACTTTGAAAC TCTCAAAGTT 360 GATTTTCTTA GCAAGCTACCTGAAATGCTG AAAATGTTCG AAGATCGTTT ATGTCATAAA 420 ACATATTTAA ATGGTGATCATGTAACCCAT CCTGACTTCA TGTTGTATGA CGCTCTTGAT 480 GTTGTTTTAT ACATGGACCCAATGTGCCTG GATGCGTTCC CAAAATTAGT TTGTTTTAAA 540 AAACGTATTG AAGCTATCCCACAAATTGAT AAGTACTTGA AATCCAGCAA GTATATAGCA 600 TGGCCTTTGC AGGGCTGGCAAGCCACGTTT GGTGGTGGCG ACCATCCTCC AAAATCGGAT 660 CTGGTTCCGC GTGGATCGGAATTCTGTTCA TTTAGTTCTA TCCCGAATGG CACGTACCGG 720 GCGACGTATC AGGATTTTGATGAGAATGGT TGGAAGGACT TTCTCGAGGT TACTTTTGAT 780 GGTGGCAAGA TGGTGCAGGTGGTTTACGAT TATCAGCATA AAGAAGGGCG GTTTAAGTCC 840 CAGGACGCTG ACTACCATCGGGTCATGTAT GCATCCTCGG GCATAGGTCC TGAAAAGGCC 900 TTCAGAGAGC TCGCCGATGCTTTGCTTGAA AAGGGTAATC CCGAGATGGT GGATGTGGTC 960 ACCGGTGCAA CTGTTTCTTCCCAGAGTTTC AGGAGGTTGG GTCGTGCGCT TCTGCAGAGT 1020 GCGCGGCGCG GCGAGAAGGAAGCCATTATT AGCAGGTAGG AATTCGTCGA CCTCGAGGGA 1080 TCCGGGCCCT CTAGATGCGGCCGCATGCAT GGTACCTAA 1119 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 352 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (A) ORGANISM: RECOMBINANT (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:15: FROM 1 TO 352 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Met SerPro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 ThrArg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 TyrGlu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 GlyLeu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 LeuThr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala LeuAsp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala PhePro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro GlnIle Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro LeuGln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro LysSer Asp Leu Val Pro Arg 210 215 220 Gly Ser Glu Phe Cys Ser Phe Ser SerIle Pro Asn Gly Thr Tyr Arg 225 230 235 240 Ala Thr Tyr Gln Asp Phe AspGlu Asn Gly Trp Lys Asp Phe Leu Glu 245 250 255 Val Thr Phe Asp Gly GlyLys Met Val Gln Val Val Tyr Asp Tyr Gln 260 265 270 His Lys Glu Gly ArgPhe Lys Ser Gln Asp Ala Asp Tyr His Arg Val 275 280 285 Met Tyr Ala SerSer Gly Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu 290 295 300 Ala Asp AlaLeu Leu Glu Lys Gly Asn Pro Glu Met Val Asp Val Val 305 310 315 320 ThrGly Ala Thr Val Ser Ser Gln Ser Phe Arg Arg Leu Gly Arg Ala 325 330 335Leu Leu Gln Ser Ala Arg Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg 340 345350 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINALSOURCE: (A) ORGANISM: E. COLI, Tp (B) STRAIN: DH15A, NICHOLS (x)PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE:FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCEAND METHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQID NO:16: FROM 1 TO 858 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:ATGTTACACC AACAACGAAA CCAACACGCC AGGCTTATTC CTGTGGAGTT ATATATGAGC 60GATAAAATTA TTCACCTGAC TGACGACAGT TTTGACACGG ATGTACTCAA AGCGGACGGG 120GCGATCCTCG TCGATTTCTG GGCAGAGTGG TGCGGTCCGT GCAAAATGAT CGCCCCGATT 180CTGGATGAAA TCGCTGACGA ATATCAGGGC AAACTGACCG TTGCAAAACT GAACATCGAT 240CAAAACCCTG GCACTGCGCC GAAATATGGC ATCCGTGGTA TCCCGACTCT GCTGCTGTTC 300AAAAACGGTG AAGTGGCGGC AACCAAAGTG GGTGCACTGT CTAAAGGTCA GTTGAAAGAG 360TTCCTCGACG CTAACCTGGC GGAGCTCGGT GGTTCTTCTC TGGTTCCGCG TGGATCGGAA 420TTCTGTTCAT TTAGTTCTAT CCCGAATGGC ACGTACCGGG CGACGTATCA GGATTTTGAT 480GAGAATGGTT GGAAGGACTT TCTCGAGGTT ACTTTTGATG GTGGCAAGAT GGTGCAGGTG 540GTTTACGATT ATCAGCATAA AGAAGGGCGG TTTAAGTCCC AGGACGCTGA CTACCATCGG 600GTCATGTATG CATCCTCGGG CATAGGTCCT GAAAAGGCCT TCAGAGAGCT CGCCGATGCT 660TTGCTTGAAA AGGGTAATCC CGAGATGGTG GATGTGGTCA CCGGTGCAAC TGTTTCTTCC 720CAGAGTTTCA GGAGGTTGGG TCGTGCGCTT CTGCAGAGTG CGCGGCGCGG CGAGAAGGAA 780GCCATTATTA GCAGGTAGGA ATTCGTCGAC CTCGAGGGAT CCGGGCCCTC TAGATGCGGC 840CGCATGCATG GTACCTAA 858 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 265 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (A) ORGANISM: RECOMBINANT (x) PUBLICATIONINFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B) TITLE: FUSED DNASEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNA SEQUENCE ANDMETHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANT RESIDUES IN SEQ IDNO:17: FROM 1 TO 265 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met LeuHis Gln Gln Arg Asn Gln His Ala Arg Leu Ile Pro Val Glu 1 5 10 15 LeuTyr Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp 20 25 30 ThrAsp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala 35 40 45 GluTrp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile 50 55 60 AlaAsp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp 65 70 75 80Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr 85 90 95Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala 100 105110 Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Glu 115120 125 Leu Gly Gly Ser Ser Leu Val Pro Arg Gly Ser Glu Phe Cys Ser Phe130 135 140 Ser Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp PheAsp 145 150 155 160 Glu Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe AspGly Gly Lys 165 170 175 Met Val Gln Val Val Tyr Asp Tyr Gln His Lys GluGly Arg Phe Lys 180 185 190 Ser Gln Asp Ala Asp Tyr His Arg Val Met TyrAla Ser Ser Gly Ile 195 200 205 Gly Pro Glu Lys Ala Phe Arg Glu Leu AlaAsp Ala Leu Leu Glu Lys 210 215 220 Gly Asn Pro Glu Met Val Asp Val ValThr Gly Ala Thr Val Ser Ser 225 230 235 240 Gln Ser Phe Arg Arg Leu GlyArg Ala Leu Leu Gln Ser Ala Arg Arg 245 250 255 Gly Glu Lys Glu Ala IleIle Ser Arg 260 265 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 672 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: SYNTHESIZED, Tp (B)STRAIN: NICHOLS (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJIIET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAIDFUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:18: FROM 1 TO 672 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:18: ATGGCGTGGA AGGTTTCTGT CGACCAAGAC ACCTGTATAGGAGATGCCAT CTGTGCAAGC 60 CTCTGTCCAG ACGTCTTTGA GATGAACGAT GAAGGAAAGGCCCAACCAAA GGTAGAGGTT 120 ATTGAGGACG AAGAGCTCTA CAACTGTGCT AAGGAAGCTATGGAGGCCTG TCCAGTTAGT 180 GCTATTACTA TTGAGGAGGC TGGTGGTTCT TCTCTGGTTCCGCGTGGATC GGAATTCTGT 240 TCATTTAGTT CTATCCCGAA TGGCACGTAC CGGGCGACGTATCAGGATTT TGATGAGAAT 300 GGTTGGAAGG ACTTTCTCGA GGTTACTTTT GATGGTGGCAAGATGGTGCA GGTGGTTTAC 360 GATTATCAGC ATAAAGAAGG GCGGTTTAAG TCCCAGGACGCTGACTACCA TCGGGTCATG 420 TATGCATCCT CGGGCATAGG TCCTGAAAAG GCCTTCAGAGAGCTCGCCGA TGCTTTGCTT 480 GAAAAGGGTA ATCCCGAGAT GGTGGATGTG GTCACCGGTGCAACTGTTTC TTCCCAGAGT 540 TTCAGGAGGT TGGGTCGTGC GCTTCTGCAG AGTGCGCGGCGCGGCGAGAA GGAAGCCATT 600 ATTAGCAGGT AGGAATTCGT CGACCTCGAG GGATCCGGGCCCTCTAGATG CGGCCGCATG 660 CATGGTACCT AA 672 (2) INFORMATION FOR SEQ IDNO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 203 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: RECOMBINANT(x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJII ET AL, (B)TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAID FUSED DNASEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K) RELEVANTRESIDUES IN SEQ ID NO:19: FROM 1 TO 203 (xi) SEQUENCE DESCRIPTION: SEQID NO:19: Met Ala Trp Lys Val Ser Val Asp Gln Asp Thr Cys Ile Gly AspAla 1 5 10 15 Ile Cys Ala Ser Leu Cys Pro Asp Val Phe Glu Met Asn AspGlu Gly 20 25 30 Lys Ala Gln Pro Lys Val Glu Val Ile Glu Asp Glu Glu LeuTyr Asn 35 40 45 Cys Ala Lys Glu Ala Met Glu Ala Cys Pro Val Ser Ala IleThr Ile 50 55 60 Glu Glu Ala Gly Gly Ser Ser Leu Val Pro Arg Gly Ser GluPhe Cys 65 70 75 80 Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr Arg Ala ThrTyr Gln Asp 85 90 95 Phe Asp Glu Asn Gly Trp Lys Asp Phe Leu Glu Val ThrPhe Asp Gly 100 105 110 Gly Lys Met Val Gln Val Val Tyr Asp Tyr Gln HisLys Glu Gly Arg 115 120 125 Phe Lys Ser Gln Asp Ala Asp Tyr His Arg ValMet Tyr Ala Ser Ser 130 135 140 Gly Ile Gly Pro Glu Lys Ala Phe Arg GluLeu Ala Asp Ala Leu Leu 145 150 155 160 Glu Lys Gly Asn Pro Glu Met ValAsp Val Val Thr Gly Ala Thr Val 165 170 175 Ser Ser Gln Ser Phe Arg ArgLeu Gly Arg Ala Leu Leu Gln Ser Ala 180 185 190 Arg Arg Gly Glu Lys GluAla Ile Ile Ser Arg 195 200 (2) INFORMATION FOR SEQ ID NO:20: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1035 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: SYNTHESIZED, Tp (B)STRAIN: NICHOLS (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKI FUJIIET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROM SAIDFUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (xi)SEQUENCE DESCRIPTION: SEQ ID NO:20: ATGAAGATTG GTATTGTAAC TGGTATCCCTGGTGTAGGGA AAAGTACTGT CTTGGCTAAA 60 GTTAAAGAGA TATTGGATAA TCAAGGTATAAATAACAAGA TCATAAATTA TGGAGATTTT 120 ATGTTAGCAA CAGCATTAAA ATTAGGCTATGCTAAAGATA GAGACGAAAT GAGAAAATTA 180 TCTGTAGAAA AGCAGAAGAA ATTGCAGATTGATGCGGCTA AAGGTATAGC TGAAGAGGCA 240 AGAGCAGGTG GAGAAGGATA TCTGTTCATAGATACGCACG CTGTGATACG TACACCCTCT 300 GGATATTTAC CTGGTTTACC GTCAGATATAATTACAGAAA TAAATCCGTC TGTTATCTTT 360 TTACTGGAAG CTGATCCTAA GATAATATTATCAAGGCAAA AGAGAGATAC AACAAGGAAT 420 AGAAATGATT ATAGTGACGA ATCAGTTATATTAGAAACCA TAAACTTCGC TAGATATGCA 480 GCTACTGCTT CTGCAGTATT AGCCGGTTCTACTGTTAAGG TAATTGTAAA CGTGGAAGGA 540 GATCCTAGTA TAGCAGCTAA TGAGATAATAAGGTCTATGA AGGGTGGTTC TTCTCTGGTT 600 CCGCGTGGAT CGGAATTCTG TTCATTTAGTTCTATCCCGA ATGGCACGTA CCGGGCGACG 660 TATCAGGATT TTGATGAGAA TGGTTGGAAGGACTTTCTCG AGGTTACTTT TGATGGTGGC 720 AAGATGGTGC AGGTGGTTTA CGATTATCAGCATAAAGAAG GGCGGTTTAA GTCCCAGGAC 780 GCTGACTACC ATCGGGTCAT GTATGCATCCTCGGGCATAG GTCCTGAAAA GGCCTTCAGA 840 GAGCTCGCCG ATGCTTTGCT TGAAAAGGGTAATCCCGAGA TGGTGGATGT GGTCACCGGT 900 GCAACTGTTT CTTCCCAGAG TTTCAGGAGGTTGGGTCGTG CGCTTCTGCA GAGTGCGCGG 960 CGCGGCGAGA AGGAAGCCAT TATTAGCAGGTAGGGATCCG GGCCCTCTAG ATGCGGCCGC 1020 ATGCATGGTA CCTAA 1035 (2)INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:330 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A)ORGANISM: RECOMBINANT (x) PUBLICATION INFORMATION: (A) AUTHORS: NOBUYUKIFUJII ET AL, (B) TITLE: FUSED DNA SEQUENCE, FUSED PROTEIN EXPRESSED FROMSAID FUSED DNA SEQUENCE AND METHOD OF EXPRESSING SAID FUSED PROTEIN (K)RELEVANT RESIDUES IN SEQ ID NO:21: FROM 1 TO 330 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:21: Met Lys Ile Gly Ile Val Thr Gly Ile Pro GlyVal Gly Lys Ser Thr 1 5 10 15 Val Leu Ala Lys Val Lys Glu Ile Leu AspAsn Gln Gly Ile Asn Asn 20 25 30 Lys Ile Ile Asn Tyr Gly Asp Phe Met LeuAla Thr Ala Leu Lys Leu 35 40 45 Gly Tyr Ala Lys Asp Arg Asp Glu Met ArgLys Leu Ser Val Glu Lys 50 55 60 Gln Lys Lys Leu Gln Ile Asp Ala Ala LysGly Ile Ala Glu Glu Ala 65 70 75 80 Arg Ala Gly Gly Glu Gly Tyr Leu PheIle Asp Thr His Ala Val Ile 85 90 95 Arg Thr Pro Ser Gly Tyr Leu Pro GlyLeu Pro Ser Asp Ile Ile Thr 100 105 110 Glu Ile Asn Pro Ser Val Ile PheLeu Leu Glu Ala Asp Pro Lys Ile 115 120 125 Ile Leu Ser Arg Gln Lys ArgAsp Thr Thr Arg Asn Arg Asn Asp Tyr 130 135 140 Ser Asp Glu Ser Val IleLeu Glu Thr Ile Asn Phe Ala Arg Tyr Ala 145 150 155 160 Ala Thr Ala SerAla Val Leu Ala Gly Ser Thr Val Lys Val Ile Val 165 170 175 Asn Val GluGly Asp Pro Ser Ile Ala Ala Asn Glu Ile Ile Arg Ser 180 185 190 Met LysGly Gly Ser Ser Leu Val Pro Arg Gly Ser Glu Phe Cys Ser 195 200 205 PheSer Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp Phe 210 215 220Asp Glu Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly Gly 225 230235 240 Lys Met Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly Arg Phe245 250 255 Lys Ser Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser SerGly 260 265 270 Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala LeuLeu Glu 275 280 285 Lys Gly Asn Pro Glu Met Val Asp Val Val Thr Gly AlaThr Val Ser 290 295 300 Ser Gln Ser Phe Arg Arg Leu Gly Arg Ala Leu LeuGln Ser Ala Arg 305 310 315 320 Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg325 330

What is claimed is:
 1. A fused DNA sequence which comprises a first DNAsequence encoding a heat-resistant ferredoxin or a heat-resistantadenylate.kinase fused directly or indirectly to the 5′ end of a secondDNA sequence encoding a selected protein or peptide in a reading frame,so that (1) the fused DNA sequence produces a protein in which theheat-resistant ferredoxin or the heat-resistant adenylate kinse isattached to the amino terminus of the selected protein or peptide whenthe fused DNA is expressed in a host cell, and (2) the fused DNA isexpressed in the host cell at a higher level as compared to acorresponding DNA which comprises the DNA sequence encoding a selectedprotein or peptide but does not comprise the DNA sequence encoding aheat-resistant ferredoxin or a heat-resistant adenylate kinase.
 2. Thesequence of claim 1, wherein the first DNA sequence encodes aheat-resistant ferredoxin.
 3. The sequence of claim 1, wherein the firstDNA sequence encodes a heat-resistant adenylate kinase.
 4. The sequenceof claim 1, wherein the first DNA sequence encodes a heat-resistantferredoxin from a Pyrococcus bacterium.
 5. The sequence of claim 1,wherein the first DNA sequence encodes a heat-resistant adenylate kinasefrom a Sulfolobus bacterium.
 6. A method of expressing a peptide orprotein comprising: (a) fusing a first DNA sequence encoding aheat-resistant ferredoxin or a heat-resistant adenylate kinase eitherdirectly or indirectly to the 5′ end of a second DNA sequence encodingthe peptide or protein to be expressed; (b) operably linking said fusedsequence of (a) in a proper reading frame to sequences which direct theexpression of said fused sequence; (c) transforming a host cell with thesequences of (b); (d) culturing said transformed host; and (e)collecting from said host the protein encoded by the sequences of (a),wherein the fused DNA is expressed in the host cell at a higher level ascompared to a corresponding DNA which comprises the DNA sequenceencoding a selected protein or peptide but does not comprise the DNAsequence encoding a heat-resistant ferredoxin or a heat-resistantadenylate kinase.
 7. The sequence of claim 6, wherein the first DNAsequence encodes a heat-resistant ferredoxin.
 8. The sequence of claim6, wherein the first DNA sequence encodes a heat-resistant adenylatekinase.
 9. The sequence of claim 6, wherein the first DNA sequenceencodes a heat-resistant ferredoxin from a Pyrococcus bacterium.
 10. Thesequence of claim 6, wherein the first DNA sequence encodes aheat-resistant adenylate kinase from a Sulfolobus bacterium.